Detection of transglucosidase-catalyzed polysaccharide synthesis on a surface in real time using surface plasmon resonance spectroscopy.
Biological systems that involve enzyme catalysis at surfaces, particularly strategically important ones that involve insoluble substrates/products such as the cell wall and the starch granule, require analyses beyond classical solution state enzymology. Using a model system, we have demonstrated the real-time measurement of transglucosidase activity on a surface using surface plasmon resonance (SPR) spectroscopy. We monitored the extension of a (partially carboxymethylated) dextran surface with alternansucrase and sucrose as a glycosyl donor. Conditions were used where surface polymer synthesis rates were a function of enzyme concentration and proportional to the extent of enzyme binding to the surface. A method to determine the turnover number of the enzyme on the surface was also developed. The presence of a new amorphous polysaccharide was observed optically, detected by lectin binding and imaged by atomic force microscopy. This surface method will have utility in a wide range of carbohydrate enzyme systems including screens.
Clé
Carla
C
Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom.
Gunning
A Patrick
AP
Syson
Karl
K
Bowater
Laura
L
Field
Robert A
RA
Bornemann
Stephen
S
D055162:Biocatalysis; D005964:Glucosyltransferases / Q000378:metabolism*; D018625:Microscopy, Atomic Force; D011134:Polysaccharides / Q000032:analysis* / Q000096:biosynthesis* / Q000737:chemistry; D020349:Surface Plasmon Resonance; D013997:Time Factors
D011134:Polysaccharides; D005964:Glucosyltransferases